The present invention relates generally to the fields of diabetes. More particularly, it concerns the identification of genes and proteins responsible for diabetes and to their inhibitors for use in therapeutics.
Insulin is the primary hormone involved in glucose homeostasis. Partial or total deficiency in insulin secretion or action leads to impaired glucose metabolism and diabetes.
Diabetes is a major cause of health difficulties in the world. Non-insulin dependent diabetes mellitus (NIDDM also referred to as Type 2 diabetes) is a major public health disorder of glucose homeostasis affecting about 5% of the general population in the United States. The causes of the fasting hyperglycemia and/or glucose intolerance associated with this form of diabetes are not well understood.
Clinically, NIDDM is a heterogeneous disorder characterised by chronic hyperglycemia leading to progressive micro- and macro-vascular lesions in the cardiovascular, renal and visual systems as well as diabetic neuropathy. For these reasons, the disease may be associated with early morbidity and mortality.
In the field of genomics, various mutations in the diabetes susceptibility genes were identified, for instance in the hepatocyte nucleotide factor genes family (HNF-1, HNF-4 and HNF-6) as described in documents WO98/11254 and WO98/23780.
The role of said genes in biochemical pathways affecting synthesis or secretion of insulin by the beta cells of Langerhans islets has been identified by the phenotype analysis of knock-out mice wherein said genes or portions thereof have been deleted from their genome.
Said knock-out mammal are thereafter used as models for the identification of new compounds or new methods of treatment which could be used for decreasing the symptoms resulting from diabetes.
It is also possible to use the corresponding identified genes which will be present in a sufficient amount in a pharmaceutical composition for the treatment and/or the prevention of said disease.
However, in this field, it exists a need for the identification of new target and biological pathways which could be used for improving the treatment and/or the prevention of diabetes.
Type 2 SH2-domain-containing inositol polyphosphate 5-phosphatase or SHIP2 is closely linked to phosphatidylinositol 3xe2x80x2-kinase and Shc/ras/MAP kinase-mediated signaling events in response to stimulation by specific growth factors.
The structure of SH2-domain containing enzymes and presenting a phosphatase catalytic activity has been already described by Pesesse et al. (1997 and 1998) and Erneux et al. (1998).
It is known that said SH2-domain containing proteins shows similarity with another known inositol polyphosphate 5-phosphatase identified as molecule SHIP1 and shows also 99% identity to a previously reported sequence (INPPL-1). INPPL-1 however, did not contain an SH2 domain.
Said new sequence will be identified hereafter as the molecule SHIP2. The other known inositol 5-phosphatase SHIP1 has been the subject of an intensive research because it may be possibly involved in negative Signalling of B immune cells and could be therefore used as target for the screening of new molecules having possibly therapeutical and/or prophylactic properties in the treatment of various immune inflammatory or allergic symptoms and diseases.
Toshiyasusasaoka et al. (Diabetes Vol. 84, No. PPA 60 (1999)(XP 000905226)) describe the cloning characterisation of a rat SHIP2 molecule that does not have the SAM domain present in human SHIP2. They show that overexpression of the SHIP2 molecule inhibits insulin-induced PKB activation by the 5-inositol phosphatase activity of SHIP2. The authors suggest that SHIP2 plays a negative regulatory role in diverse biological action of insulin and that the dual regulation of the SHC-Grb2 complex and downstream molecule of PI3-kinase provides possible mechanisms of SHIP2 molecule to participate in insulin signalling.
The international patent application WO 97/12039 describes the purification and the isolation of the nucleic acid molecules comprising a sequence encoding the SH2-containing inositol-phosphatase, a vector comprising said sequence, a cell transformed by said vector and a purified and isolated SH2-containing inositol-phosphatase molecule expressed by said cell. This document describes also antibodies directed against said protein and a method for identifying a substance capable of binding to said protein.
However, the precise functions of said SH2-domain containing inositol polyphosphate 5-phosphatase SHIP2 has not yet been identified.
The inventors have discovered unexpectedly that a knock-out mammal, preferably a knock-out mouse, comprising the partial or total deletion (heterozygously or homozygously) of said SH2-domain-containing polyphosphate inositol 5-phosphatase SHIP2 sequence is hypersensitive to insulin (severe postnatal hypoglycaemia and deregulated expression of genes involved in gluconeogenesis).
This increased sensitivity is so important that after two or three days following the birth, the homozygote (SHIP2xe2x88x92/xe2x88x92) mice die from severe hypoglycemia, and that an unpaired glucose tolerance is observed in heterozygote (SHIP2+/xe2x88x92) mice.
In vitro, the absence of one or both normal alleles of the SHIP2 gene is associated with an increased activation of PKB, an effector of PdtIns 3-kinase cascade, and of MAP kinase in response to insulin. These results provide the first direct evidence that SHIP2 is a potent negative regulator of insulin signalling in vivo, and a potential therapeutic target for the treatment of type II diabetes.
A first aspect of the present invention is related to an inhibitor of said inositol polyphosphate 5-phosphatase SHIP2 molecule, preferably a human molecule, said inhibitor being directed against said molecule and being able to reduce or block its activity or expression.
A first preferred example of said inhibitor is an anti-sense RNA (of 8 to 50 bases, preferably from 10 to 30 bases in length) constructed from the complementary sequence of the messenger RNA that can be deduced from the sequence of SHIP2 molecule complementary DNA encoding the sequence (identified hereafter as SEQ ID NO. 1 and by Erneux et al. (1998)) or its complementary strand and which specifically hybridises and inhibits its expression. Said inhibitors can also be a molecule that directly or indirectly decreases SHIP2 mRNA expression (transcription factors) or stability.
A second example of said inhibitor is a mutated SHIP2 molecule or a portion thereof of more than about 30, about 50, about 100 or about 150 amino-acids (negative dominant) that would prevent the natural activity of SHIP2 by competition of the mutated molecule to interacting proteins or receptors involved in insulin production cascade. Preferably, said mutated SHIP2 molecule or portion comprises a mutation in the following amino acid sequence: RTNVPSWCDR (SEQ ID NO: 4), especially one or more mutations, preferably of the following amino acids: S, C, N, D or R of said specific SHIP2 portion sequence. Said mutation(s) affect(s) the catalytic site of the SHIP2 molecule (phosphatase activity), as described in Erneux et al. (1998). Those mutations typically would create dominant negative effects.
Other examples of said inhibitors are substrates of said SHIP2 molecule (being an enzyme) and analogues of its substrates such as the phosphatidylinositol 3,4,5-triphosphate, the inositol 1,3,4,5-tetrakisphosphate, the inositol 1,4,5 triphosphate adenophostin or any available analogue of the inositol phosphate structure including the membrane-permeant esters or phosphatidylinositol 3,4,5-triphosphate that have been used to deliver the phosphatidylinositol 3,4,5-triphosphate across cell membranes.
Said inhibitor can be also a competitive inhibitor, such as the 2,3-biphosphoglycerate, thiol blocking agents or any protein phosphatase inhibitor such as the okadaic acid or the orthovanadate. The inhibitor can be also a specific portion of more than about 30, about 50, about 100 or about 150 amino-acids of the protein structure of said enzyme, preferably a portion comprising the SH2-domain (the first 110 amino acids of the sequence SEQ ID NO. 1, the proline-rich domain (the last 351 amino acids of the sequence SEQ ID NO. 1) or more preferred portions of said sequence which are able to compete with the molecule SHIP2 recruitment to plasma membranes, thereby inhibiting its activity. The term xe2x80x9ccompetitive inhibitorxe2x80x9d is well accepted in the art.
The inhibitor could also reduce or block the activity of the SHIP2 molecule, or reduces the expression of the SHIP2 mRNA.
The inhibitor or a pharmaceutical composition comprising an acceptable carrier or diluent or the inhibitor according to the invention, being able to reduce or block the activity of the SHIP2 molecule, increases the sensibility of a patient (including a human) to insulin and could be advantageously used in the treatment or the prevention of type II diabetes and associated diseases.
Another aspect of the present invention is related to a method, kit or apparatus for the detection of known or unknown compounds which could be used as inhibitors of said inositol polyphosphate 5-phosphatase SHIP2 molecule. Said method comprises the steps of submitting said unknown compounds to an assay based upon the analysis of SHIP2 molecule activity. Said assay, is advantageously performed on a molecular construct containing the catalytic domain of the SHIP2 enzyme (for the corresponding human SHIP2 molecule identified in the enclosed sequence SEQ ID NO. 1, this ranges from the amino acid 427 to the amino acid 729 or one or more specific portions of said catalytic domain), said construct being expressed in a micro-organism, preferably in E. coli, or in a cell line and the assay comprising the means for quantifying the hydrolysis of inositol 1,3,4,5-tetrakisphosphate and the production of inositol 1,3,4-triphosphate. The expression of SHIP2 in bacteria E. coli has been described in Pesesse et al., 1998.
The kit, apparatus and method according to the invention comprise substrates commercially available. The assay according to the invention could also be based upon a quantification of the hydrolysis of phosphatidylinositol 3,4,5-triphosphate followed by thin layer chromatography analysis. A decrease in the amount of a hydrolysis product of 5-phosphatase activity refers to 10% or greater decrease in a signal analysed by the thin layer chromatography in the presence of an inhibitor relative to its absence.
Another analysis of possible inhibitors of the SHIP2 molecule activity is based upon the inhibition of SHIP2 phosphorylation on tyrosine, because this index of activation has been shown to be crucial in the activation process and the insulin sensitive pathways. The SHIP2 molecule is purified and tested for its phosphorylation by Western blot analysis with anti-phosphotyrosine antibody which could be used also as an inhibitor according to the invention.
Another analysis of possible inhibitors of SHIP2 molecules is based upon the reduction or inhibition of SHIP2 mRNA expression, because reduced expression of SHIP2 mRNA in viva is associated with increased isulin sensitivity. SHIP2 promoter region or untranslated regions (5xe2x80x2 or 3xe2x80x2) of SHIP2 cDNA are linked with a reporter gene (luciferase, CAT, . . . ) in a plasmid vector and transfected into cells in culture. Compounds to be tested are added or not to the cells, and reporter gene activity is recorded to fond out compounds that reduce reporter gene activity, i.e. compounds that act throught the promoter, 5xe2x80x2 or 3xe2x80x2 untranslated regions of SHIP2 sequences to reduce the production of the protein. A reduction of expression refers to a 10% or greater decrease in SHIP2 mRNA level in the presence of an inhibitor relative to its absence.
A last aspect of the present invention is related to a non-human knock-out mammal comprising (homozygously or heterozygously) a partial or total deletion (in its genome) of the genetic sequence encoding the inositol polyphosphate 5-phosphatase SHIP2 enzyme, said knock-out mammal being hypersensitive to insulin and which could be used as a model for the study of diseases like severe hypoglycaemia in human newborn, or unpaired glucose tolerance in adult humans as well as the study of their treatment. Said genetic sequence encodes the inositol polyphosphate 5-phosphatase SHIP2 molecule (enzyme) identified hereafter as SEQ ID NO. 1 and described by Pesesse et al. (1997).
Said non-human knock-out mammal is preferably a knock-out mouse, obtained by techniques well-known by a person skilled in the art. Said mammal is preferably obtained by a genetic modification, a partial or total deletion in the wild type sequence through the integration of a foreigner nucleic acid sequence. Said genetically modified sequence is incorporated in a vector or electroporated and reintroduced in an embryonic stem cell (ES) for which cellular clones are selected before the integration preferably in a Swiss pseudo-gravide morula-embryo according to the technique described by Carmeliet et al. (1996), allowing thereafter the selection of mice comprising the heterozygous modification of said wild-type sequence and after crossing homozygously genetically modified mice.